Compact automated cell counter

ABSTRACT

Biological cells in a liquid suspension are counted in an automated cell counter that focuses an image of the suspension on a digital imaging sensor that contains at least 4,000,000 pixels each having an area of 2×2 μm or less and that images a field of view of at least 3 mm 2 . The sensor enables the counter to compress the optical components into an optical path of less than 20 cm in height when arranged vertically with no changes in direction of the optical path as a whole, and the entire instrument has a footprint of less than 300 cm 2 . Activation of the light source, automated focusing of the sensor image, and digital cell counting are all initiated by the simple insertion of the sample holder into the instrument. The suspension is placed in a sample chamber in the form of a slide that is shaped to ensure proper orientation of the slide in the cell counter.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/238,534, filed Aug. 31, 2009, the contents of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention lies in the field of hemocytometry and systems in generalfor the counting of biological cells suspended in fluids. The focus ofthis invention is on automated cell counting systems.

2. Description of the Prior Art

Cell counting is of interest in a variety of clinical and researchprocedures, including the counting of leukocytes and erythrocytes, whichis of value in the diagnosis of various diseases or abnormal conditionsand in the monitoring of patients that are undergoing treatment for suchdiseases or conditions. Cells can be counted manually by placing a knowndilution of a sample between optically clear plates that aresufficiently close to each other (typically with a spacing on the orderof 100 microns) to form the cells into a single layer, magnifying anarea of the layer of designated dimensions to a known magnification, andcounting the cells in the magnified area through a microscope. Manualcell counters often include a grid inscribed in the counting area tolessen the burden on the user. A description of such a grid and theprocedure for its use is found in Qiu, J., U.S. Pat. No. 7,329,537 B2,issued Feb. 12, 2008, “Micro-Pattern Embedded Plastic Optical FilmDevice for Cell-Based Assays.” Regardless of how it is done, manual cellcounting is tedious and highly vulnerable to user error. Counting iscommonly aided by using a high dilution of the sample to lessen thenumber of cells in the counting area, but the accuracy of the countingdeclines with every decrease in the proportion of cells that arecounted.

Automation of cell counting procedures has been made possible by the useof digital imaging systems. An example of such a system is ImageJ, aJava-based image processing program developed at the National Institutesof Health and reported by Collins, T. J., “ImageJ for microscopy,”BioTechniques 43 (1 Suppl.): 25-30 (July 2007). The use of ImageJ inhematology systems is reported by Gering, T. E., and C. Atkinson, “Arapid method for counting nucleated erythrocytes on stained blood smearsby digital image analysis,” J. Parasitol. 90(4): 879-81 (2004). Furtherdisclosures of automated cell counting are Chang, J. K., et al., U.S.Pat. No. 7,411,680 B2, issued Aug. 12, 2008, “Device for Counting MicroParticles,” and Chang, J. K., et al., United States Patent ApplicationPublication No. US 2006/0223165 A1, published Oct. 5, 2006, “Device forCounting Cells and Method for Manufacturing the Same.”

Automated cell counting systems themselves contain an inherentstatistical uncertainty due to what is commonly referred to as “samplingerror,” which refers to the error inherent in selecting the area inwhich the automated counting is performed. One of the limitations ofautomated cell counters that are currently available is that due to thelimitations of the optical components in the instruments, the area inwhich cells are counted is of limited size compared to the entire areaoccupied by the sample. Since this limits the number of cellsaccordingly, and the error increases with every decrease in the numberof cells being counted, the typical instrument of the prior art isconstructed with a long optical path or a large footprint (the surfacearea on a laboratory bench that the instrument consumes), or both, toachieve an acceptable level of accuracy. This presents disadvantages tothe user, particularly when the instrument is to be used in a cellculture hood.

SUMMARY OF THE INVENTION

Disclosed herein is a fully self-contained instrument for highlyaccurate cell counting with minimal user intervention as well as arelatively small footprint and limited height. A cell suspension isplaced in a consumable sample vessel whose size and dimensions can varywidely, one convenient example of which is a vessel whose outerdimensions are similar to those of a microscope slide. The vessel canthus be similar in construction and dimensions to the vessel describedin US 2006/0223165 A1 referenced above, with at least one flat, shallowinternal chamber bounded on the top and bottom by flat, optically clearwindows, which can be plastic sheets, whose spacing is close enough thatmost of the cells of the sample form a layer that is one cell deep.Appropriate inlet and vent ports can be included in the vessel to allowthe chamber to be easily and completely filled with the sample. Thevessel is then placed in the instrument where it intersects a linearoptical path. The term “linear” as used herein denotes a path with noturns or other changes in direction of the light beams other than thosecaused by lenses. The vessel enters the instrument through a slot at adesignated height in the optical path, and as described below in greaterdetail, the instrument in certain embodiments of the invention containsfeatures that automatically adjust the height of the sample for purposesof focusing the sample image. Certain embodiments contain features thatcause all instrument functions to begin operation upon the insertion ofthe sample vessel into the instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a cell counting instrument representingan example of an implementation of the concepts disclosed herein.

FIG. 2 is a diagram of the optical components of the instrument of FIG.1.

FIG. 3 is a perspective view of optical components in the interior ofthe instrument of FIG. 1.

FIG. 4 is an exploded view, in perspective, of two plates constituting asample slide for use in the instrument of FIG. 1.

FIG. 5A is a view of the upper surface of the upper plate of the sampleslide of FIG. 4. FIG. 5B is a view of the lower surface of the upperplate of the sample slide of FIG. 4.

DETAILED DESCRIPTION

The upper and lower optical windows between which the cell suspension isretained inside the sample vessel are close enough that the retainedsuspension is a thin film whose lateral dimensions, i.e., its exposedlength and width, are at least an order of magnitude greater that itsthickness. The entire exposed area (i.e., lateral dimensions) of thesample chamber or a laterally dimensioned portion thereof serves as afield of view that is projected onto a digital imaging sensor thatcontains at least about 4,000,000 (four million) pixels, or in certainembodiments from about 4,000,000 to about 10,000,000 pixels, with eachpixel being no greater than about 2×2 μm (4 μm²) in size, or from about0.5×0.5 μm (0.25 μm²) to about 2×2 μm (4 μm²) in certain embodiments,and in certain of the latter from about 1×1 μm (1 μm²) to about 2×2 μm(4 μm²). The field of view imaged by the sensor is at least about 3square millimeters, and often from about 3 mm² to about 10 mm². Acomplementary metal oxide semiconductor (CMOS) is one example of adigital imaging sensor useful for this purpose. Examples of CMOS sensorsmeeting these parameters are the OV5620 and OV5632 color imagersavailable from OmniVision, Santa Clara, Calif., USA. Other examples areavailable from Aptina Imaging, a division of Micron Technology, Inc., ofSan Jose, Calif., USA. A color digital imaging sensor can also be used.Image processing to count the cells in the image generated by the CMOSsensor can be achieved by known digital counting methods, such as thosementioned above.

The image of the sample chamber can be magnified along the optical pathby a magnification that is often within a range of from about 1.5 toabout 6, or a range of from about 1.5 to about 3, with a magnificationof about 2 as an example. This can be achieved by a two-lens achromatassembly. An example of such a lens assembly is a lens of 35-mm focallength closest to the sample, a lens of 60-mm focal length closest tothe sensor, and an aperture between the two lenses. The distance betweenthe lens nearest the sample and the sample itself in this example isthus 35 mm, and the distance between the lens nearest the sensor and theimager itself is 60 mm. The magnification of the system is the ratio ofthe focal lengths of the two lenses, which in this case is 60 mm/35mm=1.7. The two lenses can each for example be 12.5 mm in diameter, andthe aperture can be 6 mm in diameter. Lenses of other diameters andfocal lengths that will produce the same or approximately the sameresults will be readily apparent to those skilled in the art. Thefootprint of the instrument is defined as the area projected by thelarger of the instrument and its support base on a plane perpendicularto the optical path. As noted above, the instrument can be constructedwith a small footprint, particularly one that is less than 300 cm² inarea.

When a flat digital imaging sensor is used, a negative lens can bepositioned below the sensor to intercept the optical signal immediatelyand to correct the focus field curvature of the achromat lens pair. Thistype of field curvature is common in optical systems and is alsoreferred to as Petzval curvature. In an illustrative embodiment, a 6mm-diameter lens with a minus-18 mm focal length is used. The lensthickness can vary but is optimally selected to correct the curvaturewithout substantially reducing the field of view.

Illumination of the sample can be achieved with a conventional lightsource at the base of the instrument and a collimating lens between thelight source and the sample. With these components the sample isilluminated by trans-illumination without a diffuser. A preferred lightsource is a single white light-emitting diode (LED) with a fluorescentcoating. An example of such a component is LUXEON® Rebel White, part no.LXML-PWN1-0050, available from Philips Lumileds Lighting Company, SanJose, Calif., USA. An example of a collimating lens is one that is 9 mmin diameter with a focal length of 18 mm. With these dimensions andthose of the preceding paragraphs, an instrument can be constructed withthe achromat lens pair approximately 35 mm above the sample, and thesensor approximately 60 mm above the achromat lens pair. With anachromat lens pair having a thickness of approximately 13 mm, the totaldistance between the sample and the sensor can be as little as 108 mm.In general, the optical path of the instrument, i.e., defined herein asthe arrangement of the components extending from the light source to theCMOS or other digital imaging sensor, can be 20 cm or less in height. Inpreferred instruments within the scope of this invention, the opticalcomponents are mounted to the housing interior in a floating mannerusing compliant counts, to avoid damage to, or misalignment of, theoptical system upon jolts to the instrument, such as might occur whenthe instrument is dropped or mishandled, or collides with anotherinstrument or piece of equipment.

As noted above, the sample vessel, which will be referred to henceforthas a sample slide in view of its similarity in size and shape to amicroscope slide, is received in the instrument through a slot that ispositioned at a location along the optical path that is at a distancefrom the nearest lens of the achromat lens pair equal to the focallength of the lens. In its preferred embodiments, the instrument as awhole is 30 cm or less in height, and the use of a digital imagingsensor as described above that employs a large number of pixels of thesmall sizes indicated permits the instrument to be constructed with theslot at a sufficient height to allow the user to comfortably insert theslot by hand, i.e., clearing the user's hand from the table on which theinstrument rests. The slot can thus be 60 mm or more from the base ofthe instrument, and preferably 70-80 mm from the base.

In preferred embodiments of the invention, the instrument providesautofocusing of the sample image by automatically adjusting the heightof the slide following its insertion. One means of autofocusing involvesthe use of an image processor chip that provides an output of imagecontrast within an array of zones across the image from the sensor. Anexample of such a chip is the Freescale Semiconductor MC9328MX21,available from Keil™—an ARM Company, Plano, Tex., USA; other exampleswill be apparent to those skilled in the art. The sum of the absolutedifferences of adjacent green pixels in a particular zone of the sensorarray can be used as the image contrast value, and optimum focus isachieved when the image contrast value is at a maximum. The focus canthen be adjusted by a geared motor connected to the slide mount withinthe receiving slot, i.e., the motor when rotated will move the slidemount up or down to change the focus of the image. The contrast value isdetected at various positions of the motor which is then returned to theposition producing the highest contrast value. In many embodiments ofthe instrument, this autofocusing can occur in 15 seconds or less.

An accessory that can be supplied with the instrument is a standardslide for quality control, such as verifying the accuracy of countinglive and dead cells and the ability of the instrument to focus properly.The standard slide can have the same external dimensions as a sampleslide, but instead of a sample chamber(s), the standard can have anarray of dark-colored spots and rings printed on it, the spotssimulating dead cells and detected as such in the digital imaging sensorand the rings simulating live cells and detected as such in the digitalimaging sensor.

In certain embodiments of the concepts described herein, the functionsperformed by the instrument, including autofocusing and cell counting,are initiated by the simple insertion of the sample slide. Thisinitiation can be achieved by the inclusion of a non-contact opticalreflection sensor located within the slot or on the slide mount withinthe slot. An example of a suitable sensor is one that emits an infra-redbeam and detects objects within approximately one millimeter of thesensor aperture by detecting a reflected signal from the beam. Thereflected signal will rise to a maximum level when the slide is fullyinserted, and the high signal will initiate the autofocusing and cellcounting mechanisms. One example of a sensor that can serve this purposeis the QRE1113 Reflective Object Sensor, available from FairchildSemiconductor Corporation, San Jose, Calif., USA. Other examples will beapparent to those skilled in the art.

A further feature that can be included in instruments embodying thefeatures described herein is the automatic detection of cells in thesample that are stained with a vital stain. A vital stain is one thatpreferentially stains dead cells, and the differentiation between cellsstained with such a stain and those that are not is achieved by the useof differently colored pixels. Trypan blue is one example of a vitalstain; eosin and propidium iodide are other examples. Trypan bluetransmits blue light and attenuates red light, and by comparing theintensities of blue and red pixels in the image sensor, the instrumentcan determine whether cells stained with a vital stain are present.Other dyes will afford similar color distinctions as appropriate to thedyes themselves. Image processing chips that incorporate this automaticdetection feature include those referenced above and are readilyavailable. The instrument can be programmed to eliminate any possibleundercounting of viable cells and thereby detect viable cells to aparticularly high degree of accuracy by focusing on two or more planes.The contrast between live cells and dead cells can be increased furtherby using optical filters to control the illumination bandwidth, or byselecting a spectrally narrow light source, such as an LED of aparticular color instead of white. For example, a 585 nm optical filterwith about 20 nm bandwidth can be used to match the illumination to thepeak absorption wavelength of the Trypan blue dye, whose peak absorptionis 586 nm. The dead cells will appear darker when the sample isilluminated through this filter.

In preferred instruments within the scope of this invention, allfunctions that contribute to the obtainment of a cell count in thesample are contained within the instrument housing, and the fulloperation of the instrument can thus be achieved without the use of anexternal machine or computer. Included among these functions are theautomatic focusing by varying the height of the sample slide to find thebest focal plane to discriminate cells from background, thedetermination of whether the sample has been stained with Trypan blue orother vital stain, a multi-focal plane analysis when a vital stain isdetected so that each cell is scored on multiple focal planes to preventundercounting of live cells, an integrated dilution counter to determinethe volume of a cell suspension to use, the ability to produce a visualimage of the cells on the display at the option of the user and to zoomin for a detailed visual inspection of the cells, and the ability anduser option to export the results to a USB flash drive or to a thermalprinter or other external printer. All of these functions can beinitiated by the simple insertion of the sample slide by way of thenon-contact optical reflection sensor described above, and in manycases, the execution of these functions is completed in 30 seconds orless.

The Figures hereto depict an instrument that contains many of thefeatures described above and serves as one example of an implementationof the concepts described herein.

FIG. 1 depicts an automated cell counter instrument 11 in its uprightposition as it would be used on a laboratory bench. The visible parts ofthe instrument are a housing 12, a support base 13, a display screen 14,a control panel 15, and a slot 16 for insertion of a sample slide 17.The display screen shows the progress of the cell counting analysis,identifies the functions of the instrument as they are being performed,and offers options to the user for various functions and for showing animage of the cells in the sample slide.

FIG. 2 depicts components of the optical path in the interior of theinstrument of FIG. 1 with the sample slide 17 having been positioned inthe optical path. The sample slide 17 is horizontal and resides above anLED board 22 serving as the light source. A collimating lens 23 rendersthe light rays from the LED parallel as they approach the sample slide.The achromat lens pair 24 is positioned between the sample slide 17 andthe sensor 25. The two lenses 26, 27 of the achromat lens pair areseparated by an aperture 28. A field flattening lens 29 is positionedimmediately below the sensor 25.

FIG. 3 depicts the main optics assembly, showing the slide mount 31 withthe sample slide 17 partially inserted, the LED board 22, theillumination (collimating) lens 23, the geared motor 32 that adjusts theslide height to focus the image, and an imaging lens tube 33 terminatingin a fitting 34 to receive the CMOS sensor board. Also shown in theFigure is the main printed circuit board 35 that controls the functionsof the instrument and includes a motor drive chip to control the motor32. The board 35 resides within the housing and the position of theboard in the Figure reflects its position relative to the opticsassembly,

A sample slide for use in the instrument of the preceding Figures isshown in FIGS. 4, 5A, and 5B. The view in FIG. 4 is a perspective view,and the slide 17 is formed of two plates 42, 43 bonded together butshown separated in the Figure. The slide contains two sample chambers,as indicated by the indicia “A” and “B,” respectively, separated fromeach other lengthwise along the slide and laterally offset from eachother. The areas 44, 45 of the lower plate 43 that form the bottomsurfaces of the sample chambers are made of optically transparentmaterial, as are the corresponding areas of the upper plate 42 that aredirectly above these areas on the lower plate and form the uppersurfaces of sample chambers. The lower plate 43 in this embodiment isthicker than the upper plate 42 to provide rigidity to the slide, andthe relative thinness of the upper plate 42 permits the upper window ofeach sample chamber to be thinner than the lower window, and indeed asthin as possible to achieve a highly focused image in the CMOS sensor.Each sample chamber is thus offset from the center plane of the slideand closer to the upper plate 42 than to the lower plate 43.

FIGS. 5A and 5B are planar views of the top surface 51 and bottomsurface 52, respectively, of the upper plate 42, the bottom surface 52being the surface that is bonded to the lower plate 43. Each samplechamber is defined by a recess 53 (FIG. 5B) in the bottom surface of theupper plate, which further reduces the thickness of the area forming theoptically clear window at the top of each sample chamber. In one exampleof the dimensions of the slide, the thickness of the upper plate inareas other than the recess 53 is 0.65 mm and the thickness of the lowerplate is 1.00 mm, for a total slide thickness of 1.65 mm. The recess 53is 0.100 mm in depth, which thus forms a sample chamber that is 0.100 mmin depth, a standard sample chamber thickness for manual hemocytometers.Each sample chamber has two loading or vent ports 54, 55, one at each ofthe two opposing longitudinal ends of the elongated chamber. Overflowareas 56, 57, 58, 59 that are open at the top of the slide arepositioned at each of the four corners of each sample chamber toaccommodate excess sample and thereby insure that the sample chamber isproperly filled with sample.

Since each sample chamber is closer to the upper plate 42 than to thelower plate 43, the slide functions best when properly inserted into thecell counter with the upper plate 42, and hence the thinnest opticalwindow, at the top. To ensure that the slide is inserted in thisorientation, the slide is formed with notches 61, 62 in two diagonallyopposing corners of the slide. The internal surfaces of the slot in thecell counter into which the slide is inserted to initiate the functionsof the cell counter contains contour features that are complementary tothese notches. The notches and complementary contours in the slotthereby prevent the user from inserting the slide upside down, i.e.,with the upper plate 42 at the bottom rather than the top. Thesymmetrical arrangement of the notches also complements the symmetricalarrangement of the two sample chambers and permits the slide to beinserted with either end first, while preventing the slide from beinginserted in an inverted position (upside down). Since the slide ispreferably a consumable item, it can thus be used for cell countingmeasurements on two independent samples at different times, and onceboth chambers have been used the slide can be disposed of and not usedagain.

Variations on the construction of the sample slide that still ensureproper orientation will be readily apparent to those skilled in the art.The arrangement, number, and shapes of the notches can thus be varied,as can the number of sample chambers and their locations relative toeach other in the slide. The material of construction can vary widelyand can be any material that can form an optically clear window, that isinert to the sample, and that is sufficiently rigid to be inserted intothe cell counter. Poly(methyl methacrylate) and polycarbonate areexamples of materials that are can be used. Others will be readilyapparent to those skilled in the art. Likewise, the bonding of theplates can be accomplished by conventional means. Laser welding andultrasonic welding are examples.

In the claims appended hereto, the terms “a” and “an” are intended tomean “one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

1. An automated cell counter comprising optical components, a samplemount for receiving a sample slide, and a housing retaining said opticalcomponents and sample mount, said optical components comprising a lightsource, a digital imaging sensor, and lenses positioned to direct lightfrom said light source to said digital imaging sensor through saidsample mount, said optical components arranged in a linear optical pathand arranged such that when a sample slide containing a cell suspensionis mounted in said sample mount, an image of said cell suspension isprojected onto said digital imaging sensor, said digital imaging sensorcomprising at least about 4,000,000 pixels and imaging a field of viewat said sample mount of at least 3 mm², each pixel being 2×2 μm or lessin area.
 2. The automated cell counter of claim 1 wherein said digitalsensor contains from about 4,000,000 to about 10,000,000 pixels, eachpixel is from about 0.5×0.5 μm to about 2×2 μm in area, and said imagingfield is from about 3 mm² to about 10 mm².
 3. The automated cell counterof claim 1 wherein said optical path is vertically oriented and saidhousing is mounted to a support base that is at least as wide as saidhousing, said housing and support base each occupying lateral areas ofnot more than 300 cm² and said optical path having a total height of notmore than 20 cm.
 4. The automated cell counter of claim 1 wherein saiddigital imaging sensor comprises means for generating contrast valuesbetween adjacent regions of said image, said sample mount is ofadjustable height along to said optical path, and said cell counterfurther comprises means for automated adjustment of said height inresponse to said contrast values until said image is focused.
 5. Theautomated cell counter of claim 4 further comprising a digital imagerthat counts cells in said field of view.
 6. The automated cell counterof claim 5 wherein said automated adjustment of said height of saidsample mount, and digital imager, are initiated automatically uponinsertion of a sample slide in said sample mount.
 7. The automated cellcounter of claim 1 wherein said digital imaging sensor is flat, saidlenses comprise an achromat lens pair having a focus field at saiddigital imaging sensor, and said automated cell counter furthercomprises means for correcting curvature of said focus field.
 8. Theautomated cell counter of claim 7 wherein said means for correctingcurvature of said focus field is a negative lens adjacent to saiddigital imaging sensor.
 9. The automated cell counter of claim 1 whereinsaid light source is spectrally limited to produce an image in saidimaging sensor that differentiates between live cells and dead cells.10. The automated cell counter of claim 1 wherein said digital imagingsensor is a color digital sensor.
 11. The automated cell counter ofclaim 3 wherein, when said support base rests on a surface, said housingis 30 cm or less in height and said sample mount is at least 6 cm abovesaid surface.
 12. The automated cell counter of claim 1 furthercomprising a quality control slide dimensioned to be mounted on saidsample mount and having images printed thereon to simulate live cellsand dead cells.
 13. A sample slide for counting cells in a cellsuspension, said sample slide comprising: a flat plate with an internalchamber bounded by optically clear upper and lower windows, and anorientation notch in said flat plate to guide said flat plate into acell counting instrument in an orientation whereby said upper windowfaces a selected direction within said instrument.
 14. The sample slideof claim 13 wherein said upper window is substantially thinner than saidlower window.
 15. The sample slide of claim 13 comprising a single flatplate with first and second internal chambers therein, said internalchambers positioned laterally relative to each other in said flat plateand each having optically clear upper and lower windows, said singleflat plate comprising first and second orientation notches to guide saidflat plate into said cell counting instrument in orientations wherebysaid optically clear upper windows face a selected direction within saidinstrument.
 16. A method for counting cells in a cell suspension, saidmethod comprising: (a) placing an aliquot of said cell suspension in aninternal chamber of a sample slide to form a film of said suspension insaid chamber, (b) creating a digital image of a field of view of atleast 3 mm² within said film on a digital imaging sensor comprising atleast 4,000,000 pixels, each pixel being 2×2 or less in area, byinserting said sample slide on a sample mount and positioning saidsample mount in a linear optical path formed of optical componentscomprising a light source, said digital imaging sensor, and lensespositioned to direct light from said light source to said digitalimaging sensor through said sample mount, and (c) digitally countingsaid cells in said digital image.
 17. The method of claim 16 whereinsaid digital sensor contains from about 4,000,000 to about 10,000,000pixels, each pixel is from about 0.5×0.5 μm to about 2×2 μm in area, andsaid imaging field is from about 3 mm² to about 10 mm².
 18. The methodof claim 16 wherein step (b) is performed by a plurality of functionscomprising automatic focusing of said digital image by moving saidsample mount, detecting whether a vital stain is present in saidaliquot, creating digital images of a plurality of focal planes withinsaid internal chamber of said sample slide when a vital stain isdetected, and processing images formed on said digital imaging sensor todetermine a cell count in said aliquot, are all performed internallywithin said housing.
 19. The method of claim 16 wherein said opticalpath is vertically oriented and said sample mount and said opticalcomponents are retained in a housing mounted to a support base that isat least as wide as said housing, said housing and support base eachoccupying lateral areas of not more than 300 cm² and said optical pathhaving a total height of not more than 20 cm.
 20. The method of claim 16further comprising (a′) automatically generating contrast values betweenadjacent regions of said image, and automatically adjusting said samplemount to a height along said optical path in response to said contrastvalues to focus said image.
 21. The method of claim 16 wherein steps(a′) and (b) are initiated automatically upon insertion of said sampleslide in said sample mount.
 22. The method of claim 16 wherein saiddigital imaging sensor is flat, and said lenses comprise an achromatlens pair having a focus field at said digital imaging sensor and anegative lens positioned to correct curvature of said focus field. 23.The method of claim 16 wherein said light source is spectrally limitedto produce an image in said imaging sensor that differentiates betweenlive cells and dead cells.
 24. The method of claim 16 further comprisingtreating said cell suspension with a vital stain and thereby producingan image in said imaging sensor that differentiates between live cellsand dead cells.
 25. The method of claim 16 wherein said digital imagingsensor is a color digital sensor.
 26. The method of claim 16 whereinstep (c) comprises focusing said optical components on a plurality ofplanes within said internal chamber.